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Open AccessResearch Proteomic characterization of HIV-modulated membrane receptors, kinases and signaling proteins involved in novel angiogenic pathways Suraiya Rasheed*, Jasper S Yan,

Open Access

Research

Proteomic characterization of HIV-modulated membrane

receptors, kinases and signaling proteins involved in novel

angiogenic pathways

Suraiya Rasheed*, Jasper S Yan, Adil Hussain and Bruce Lai

Address: Laboratory of Viral Oncology and Proteomics Research Department of Pathology, Keck School of Medicine, University of Southern

California, 1840 N Soto St, Los Angeles, CA 90032-3626, USA

Email: Suraiya Rasheed* - srasheed@usc.edu; Jasper S Yan - Jasper.S.Yan@rice.edu; Adil Hussain - ahussain@uci.edu; Bruce Lai - bpl@duke.edu

* Corresponding author

Abstract

Background: Kaposi's sarcoma (KS), hemangioma, and other angioproliferative diseases are highly prevalent in

HIV-infected individuals While KS is etiologically linked to the human herpesvirus-8 (HHV8) infection, HIV-patients without

HHV-8 and those infected with unrelated viruses also develop angiopathies Further, HIV-Tat can activate

protein-tyrosine-kinase (PTK-activity) of the vascular endothelial growth factor receptor involved in stimulating angiogenic

processes However, Tat by itself or HHV8-genes alone cannot induce angiogenesis in vivo unless specific proteins/

enzymes are produced synchronously by different cell-types We therefore tested a hypothesis that chronic

HIV-replication in non-endothelial cells may produce novel factors that provoke angiogenic pathways.

Methods: Genome-wide proteins from HIV-infected and uninfected T-lymphocytes were tested by subtractive

proteomics analyses at various stages of virus and cell growth in vitro over a period of two years Several thousand

differentially regulated proteins were identified by mass spectrometry (MS) and >200 proteins were confirmed in multiple

gels Each protein was scrutinized extensively by protein-interaction-pathways, bioinformatics, and statistical analyses

Results: By functional categorization, 31 proteins were identified to be associated with various signaling events involved

in angiogenesis 88% proteins were located in the plasma membrane or extracellular matrix and >90% were found to be

essential for regeneration, neovascularization and angiogenic processes during embryonic development

Conclusion: Chronic HIV-infection of T-cells produces membrane receptor-PTKs, serine-threonine kinases, growth

factors, adhesion molecules and many diffusible signaling proteins that have not been previously reported in HIV-infected

cells Each protein has been associated with endothelial cell-growth, morphogenesis, sprouting, microvessel-formation

and other biological processes involved in angiogenesis (p = 10-4 to 10-12) Bioinformatics analyses suggest that

overproduction of PTKs and other kinases in HIV-infected cells has suppressed VEGF/VEGFR-PTK expression and

promoted VEGFR-independent pathways This unique mechanism is similar to that observed in neovascularization and

angiogenesis during embryogenesis Validation of clinically relevant proteins by gene-silencing and translational studies in

vivo would identify specific targets that can be used for early diagnosis of angiogenic disorders and future development

of inhibitors of angiopathies This is the first comprehensive study to demonstrate that HIV-infection alone, without any

co-infection or treatment, can induce numerous "embryonic" proteins and kinases capable of generating novel

VEGF-independent angiogenic pathways.

Published: 27 August 2009

Journal of Translational Medicine 2009, 7:75 doi:10.1186/1479-5876-7-75

Received: 1 April 2009 Accepted: 27 August 2009 This article is available from: http://www.translational-medicine.com/content/7/1/75

© 2009 Rasheed et al; licensee BioMed Central Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Angiogenesis, or the formation of new blood vessels from

the existing ones, is an essential biological process for

maintaining numerous physiological functions ranging

from cell growth, proliferation, repair of damaged cells to

wound-healing in vivo [1-3] Throughout the life of an

individual and during embryonic development, various

pro-angiogenic and anti-angiogenic factors (i.e

promot-ers and inhibitors of angiogenesis respectively) produced

by various cell types maintain a balance between

neovas-cularization and angiogenesis programs in a cyclic

man-ner [4,5] Exactly how abnormal angiogenic signals are

generated in vivo is not well-understood, but an imbalance

in the production of one or more critical factors can alter

the protein-protein interaction pathways and induce

ang-iogenic anomalies including inflammation, vascular

dementia, hemangioma, dysfunctional uterine bleeding,

ovarian hyperstimulation and choroidal/intraocular

dis-orders to name a few [1,6] Angiogenesis is also critical for

cancer metastasis, diabetic blindness, age-related macular

degeneration, rheumatoid arthritis, psoriasis, and for the

development of new blood vessels that supply oxygen and

nutrients to the body when aortas are clogged

(thrombo-sis) [2,6]

In both the neoplastic and non-neoplastic diseases,

endothelial cells have been shown to express various

iso-forms of the vascular endothelial growth factors (VEGFs)

which bind to their cognate VEGF receptors (VEGFRs),

activate their associated protein tyrosine kinases (PTKs)

and stimulate endothelial cell growth through angiogenic

pathways [3,6,7] However, endothelial cells can be

acti-vated by various cytokines, phosphorylated proteins and

other factors that are essential not only for cell growth but

also for maintaining an activated state of the stimulated

endothelial cells [2,8] In the absence of specific cytokines

and diffusible signaling proteins, VEGF by itself is not

suf-ficient to trigger expression of numerous enzymes and

proteins required for the development of a network of

blood vessels from the existing vasculature [8,9]

Angiogenic Factors are also produced by Pathogenic

Viruses

Etiologic factors involved in different types of

vasculopa-thies in humans have not been fully explored However,

in the absence of any tumor growth many DNA or RNA

viruses have been shown to cause vascular lesions in vivo

or produce proangiogenic factors in vitro For example, the

human herpes simplex virus type 1 (HSV-1)-infected

ocu-lar cells produce IL-6, which stimulates uninfected,

avascu-lar corneal cells to secrete VEGF and provoke

neovascularization in the eye [10] Infection with the

Epstein-Barr virus (EBV) enhances production of many

cytokines and causes angiogenic cutaneous tumors [11]

The dengue virus, causes hemorrhagic fever and vascular

lesions in humans, produces interleukin-4 (4), 8,

IL-6, IL-10, GM- colony stimulating factor (CSF), gamma (INF-gamma) and tumor necrosis factor alpha(TNF-alpha) [12] The human parapoxvirus causes exten-sive skin vasculopathies and the pseudocowpox viral

interferon-genome induces viral gene-encoded VEGF homologues (i.e.

VEGF-like factors) [13,14] Likewise, the common humanrhinovirus infection produces factors that promote angio-genesis in bronchial epithelial cells [15]

One of the best-studied models of angiogenesis is Kaposi'ssarcoma (KS), a highly vascular tumor that is rare in thegeneral population but occurs frequently in humanimmunodeficiency virus (HIV)-infected individuals [16-18] However, KS is etiologically associated with thehuman herpesvirus-type-8 (HHV-8) infection sinceHHV8-genome itself encodes a viral G-protein-coupledreceptor (vGPCR), which activates both oncogenic and

angiogenic pathways in the presence or absence of

HIV-coin-fection [17,19,20].

Many HIV-infected patients, who may or may not beinfected with HHV8, develop intraepithelial neoplasia,hemangiomas, lymphomas, angiosarcomas, myelodys-plastic angiogenic syndrome and other angiopathies [21-23] The HIV-encoded transcriptional transactivator (Tat)protein has been implicated in angiogenesis because itbinds VEGFR and stimulates endothelial cell growth [17].However, its binding-affinity is not as strong as that of the

natural cellular VEGFs and the avidity of Tat interaction with VEGFR is dependent on specific cytokines produced

locally by endothelial cells, cancer cells or other

virus-infected and unvirus-infected cell types in vivo [10,13,24,25] Further, the activated state of endothelial cells must be main-

tained continuously during the numerous biological

proc-esses that lead to angiogenesis These data suggest that

while Tat synergizes the effects of many viral and cellular

factors during the complex biological processes of genesis, Tat alone or individual cytokines by themselves

angio-do not induce angiogenesis in mice

The molecular mechanisms involved in HIV-induced culopathies in humans are difficult, if not impossible tostudy because most patients are co-infected with differentpathogenic viruses such as HSV-1, HSV11, EBV, hepatitis

vas-B virus (Hvas-BV), hepatitis C virus (HCV), human papillomavirus (HPV) and different bacterial and fungal microor-ganisms Consequently, cellular changes induced by HIV

alone in vivo can not be distinguished from those

pro-duced by other viruses or pathogenic organisms iting the same individual, unless separate protein profiles

co-inhab-of each class co-inhab-of different infectious agents are established

first We therefore tested a hypothesis that chronic

HIV-rep-lication in non-endothelial cells induces novel cellular

pro-teins that provoke specific protein-protein interactions

along the angiogenic pathways Although most in vitro

studies have utilized endothelial cells derived from early

KS lesions or human veins (by necessity), in this study we

preferred to use T-cells because some differentiated

endothelial cells may already produce proangiogenic

cytokines in response to changes in the cellular milieu or

alternatively, factors that are essential for endothelial cell

activation may be experimentally induced [26,27]

Herein, we report that HIV- infected human T-cells

pro-duce numerous kinases, adhesion molecules and other

angiogenic factors (not encoded by HIV-genome) that are

capable of initiating and promoting novel

VEGF-independ-ent pathways These mechanisms are similar to those

observed during embryonic development,

neovasculari-zation and angiogenesis

Experimental design and methods

To identify possible factors that can be associated with

HIV-infection alone, we used a single-cell-cloned human

T-cell line (RH9) consisting of a homogeneous

popula-tion of cells [28] These cells are highly susceptible to the

replication of most global HIV-strains tested including

those that are preferentially

"macrophage/monocyte-tropic" (SR personal observation) The RH9 cells do not

induce cytopathic effects but occasionally, when some

chronically infected cultures exhibit syncytia, uninfected

counterpart cells are added to maintain long-term

HIV-infected cell lines

The choice of T-cells for HIV infection was also based on

the fact that T-cells, together with monocytes and

macro-phages present at the portal of entry in vivo are the first cell

types to be infected soon after HIV-exposure Our

experi-ments were deliberately designed to avoid the use of

pri-mary T-cells for HIV-infection due to the genetic

heterogeneity and sample-to-sample variation in the

sus-ceptibility of freshly cultured human peripheral blood

mononuclear cells (PBMC) (SR unpublished data) Since

HIV-infected individuals harbor a variety of different

strains (present as quasispecies in vivo), we used a

biolog-ically cloned HIV strain (X4) in order to have better

repro-ducibility and consistency of results from experiment to

experiment This methodology reduced variations in their

replication potentials

While several HIV-infected T-cell lines or Tat-transfected

T-cell lines have been used to study HIV-infected

pro-teomes and gene expression profiles, all of these analyses

were conducted after a short time (24–48 hrs) of infection

or transfection of cells [29-32] Given that most

HIV-dis-eases including vasculopathies are developed after several

years of chronic infection, we compared genome-wide

proteins from HIV-infected and counterpart uninfected

T-lymphocytes over a period of two years by subtractive

pro-teomics, bioinformatics and statistical analyses These

studies were designed to evaluate only the differentially regulated (i.e upregulated, downregulated or de novo induced proteins post-HIV infection), and not the entire

proteome of the HIV-infected or uninfected cells Finally,

all experiments were conducted in the absence of other

pathogenic viruses or microbes that may produce iogenic factors

proang-Virus Infection for Proteomics Studies

Approximately 109 cells were plated in each of the twolarge flasks at a density of 2 × 106 cells per ml in RPMI

1640 medium supplemented with 20% fetal bovineserum (FBS), 2 mM glutamine and 2 μg/ml polybrene.After 16–18 hours (h), one culture was infected with HIV

at a multiplicity of infection of one (MOI = 1) and bothinfected and uninfected cultures were incubated at 37°C

in an atmosphere of 5% CO2 After 1.5 h, all cells fromboth flasks were harvested separately, washed with phos-

phate buffered saline (PBS) and transferred to new flasks

with fresh medium without polybrene

Numerous experiments were conducted over a period ofmore than two years and changes in protein profiles wereanalyzed in relation to various HIV-associated dysfunc-tions/diseases One experiment was conducted forapproximately 3 months and duplicate samples fromHIV-infected and counterpart uninfected samples weretested at 14 time points by proteomics analyses Thesesamples ranged from 1.5 h to 96 days (d) post-infection(3 h, 6 h, 12 h, 24 h, 48 h, 4 d, 10 d, 14 d, 20 d, 26 d, 28

d, 47 d and 96 d) In subsequent experiments, sampleswere harvested at the peak of HIV-replication (i.e from 10

to 26 days) Given that most HIV-associated diseasesdevelop after a chronic infection, we tested an additionalten different chronically HIV-infected and uninfectedcounterpart cells selected randomly over a period of twoyears i.e at various stages of virus replication and cellgrowth This large sample size was necessary in order toselect highly reproducible protein spots in multiple gelsand for testing many quality-control samples used forstandardization of experiments such as lyophilized E coliextract, commercially available purified proteins and asingle extract of HIV-infected and uninfected cells

Isolation of Plasma Membrane and Extracellular Matrix Proteins

A major goal of this study was to identify cell surface teins involved in generating HIV-modulated signals thatdisrupt normal cellular functions and drive infected cells

pro-in specific directions Over the years our laboratory hasdeveloped a rapid sequential extraction procedure to suc-cessfully isolate functionally relevant and naturally occur-ring plasma membrane and extracellular matrix proteins[33,34] All proteins were isolated by unbiasedapproaches (i.e without the use of special ligands, anti-

bodies or ion exchange columns or liquid

chromatogra-phy for capturing or purifying specific proteins) Although

this may not be an ideal method for identifying the entire

proteome, this method was excellent for identifying many

differentially expressed signal transduction molecules

Briefly, aliquots of 107 cells from each of the HIV- infected

and uninfected cultures were removed at various time

points as indicated above, and washed with PBS by low

speed centrifugation twice and once with normal saline

(0.9% NaCl) The cell pellets were lysed rapidly for 15

sec-onds using (8 M Urea, 2% (w/v) CHAPS, 2%

mercap-toethanol, 2.5% protease inhibitor cocktail, and 150

units/200 μl endonuclease) Each lysate was then vortexed

gently and sonicated for 2 seconds followed by

centrifuga-tion at 14,000 rpm for 10 minutes Just before loading the

gels, the clarified supernatant from the lysate was

centri-fuged again at 100,000 × g for 90 minutes in a high-speed

centrifuge and processed for protein fractionation by

two-dimensional gel electrophoresis All proteins were

sepa-rated first by isoelectric focusing on various pH gradients

(3 to10) and size fractionated in the second dimension by

gel electrophoresis on gradient polyacrylamide gels (6–

18%)

Electrophoretically separated proteins in the gels were

washed 3× with double-distilled H2O and stained with

Coomassie Brilliant Blue for 30 minutes and de-stained in

15% (v/v) methanol, 7% (v/v) acetic acid for a minimum

of three hours Several Coomassie-stained gels were

coun-terstained with Sypro Ruby Red (SRR) fluorescent dye

after the gels were scanned for image-analysis and double

stained gels were scanned again Since fluorescent signals

of SRR are photostable and comparable to Cy3 and Cy5

dyes [35], this procedure enhanced the sensitivity of some

light-colored spots and reduced non-specific spot identity

Bioinformatics and Statistical Analyses for Identification

of Angiogenic Proteins

Genome-wide protein profiles of both the infected and

uninfected counterpart cells were compared and

evalu-ated by subtractive proteomics analyses overtime i.e at

different stages of virus and cell growth Only those

pro-teins that were clearly identified by Matrix Assisted Laser

Desorption Ionization-Time of- Flight (MALDI-TOF)

mass spectrometry (MS) in multiple gels were included in

the final analyses Further, any "new" proteins (i.e

hypo-thetical proteins) identified by MS or peptide

fingerprint-ing with low Molecular Weight Search (MOWSE) Scores

(p = 0.05 or more) in any gel were excluded from the

cur-rent analyses regardless of the intensity of the stain

All protein profiles from the HIV-infected and uninfected

cells were compared and analyzed by a variety of

subtrac-tive computer-based approaches Integrated programs for

accuracy analyzed all proteins by calculating means and

standard deviations for quantitative evaluations of teins in both HIV-infected and uninfected controls Toidentify HIV-modulated proteins related to angiogenesis,

pro-we have used several bioinformatics programs and gene/protein databases including the Online Mendelian Inher-itance in Man (OMIM), a database of human genes andgenetic disorders The Ingenuity Pathway Analyses (IPA)Systems and Computational Biology programs were used

to analyze global canonical and protein-interaction ways for each of the identified proteins Each protein wasalso functionally categorized to identify possible roles inthe numerous stepwise processes, from HIV-induced cellactivation to the formation of a network of new blood ves-sels from the existing endothelial cells

path-Each differentially regulated protein was analyzed for itsbiological significance relative to those present in the glo-bal gene/protein databases available in the publicdomains and cell type-specific functionality by the use ofIngenuity-IPA/computational programs The numbers of

"focus" proteins (Table 1) were annotated in relation tothe total number of genes/proteins known to be associ-ated with various essential biological processes involved

in endothelial cell growth, formation of blood vessel andother categories recorded in the Ingenuity's knowledge-base The p-values were calculated using IPA and the right-tailed Fisher Exact Test for each of the various biological/cellular processes involved in angiogenesis All p-valueswere less than 0.0001 (Table 1)

Protein-Protein Interaction Pathway Analyses

The Ingenuity Pathway Analyses (IPA) Systems and thedirect Interaction Function Bioinformatics Programs ofStratagene Pathway Architect 2.0.1 were used to analyzeprotein-protein interaction pathways All dysregulatedproteins were uploaded and function-specific pathwayswere generated automatically by using IPA as well as Strat-agene Architect programs Although similar pathwayswere constructed by the two programs, the protein-pro-tein interaction pathways presented herein were made bythe Stratagene Architect program

Results and discussion

Cell culture supernatants from all experimentally infected cells showed an exponential increase in the p24antigen levels tested over time by the enzyme-linkedimmunoassays Although many HIV-encoded proteins(gag-p24, Tat, Rev, Vpu, Vpr, Vif, gp120, gp41 and thepolymerase) were identified by mass spectrometry (MS)

HIV-in various proteHIV-in-complexes, HIV-in this study we have

focused on the identification of HIV-modulated cellular proteins only (i.e not encoded by viral genes).

Functional Categorization of Cellular Proteins

Comprehensive MS analyses of several thousand proteins

confirmed more than 200 proteins from multiple gels run

at different phases of cell growth and virus replication

over time Results presented herein have been

consoli-dated from proteomics data generated over a period of >

2 years Each of the differentially regulated proteins was

functionally categorized by the use of bioinformatics

pro-grams that integrated biological information currently

located in several global databases including Ingenuity

Systems' knowledgebase of the Functional Repository of

Human Genes We have identified 31 proteins that have

been deemed essential for numerous molecular functions

involved in neovascularization (i.e formation of blood

vessels de novo in the embryo) or in angiogenesis (i.e

gen-eration of new blood vessels from the existing

vascula-ture) Full name, abbreviation and accession number for

each protein are listed according to the information

avail-able on the latest Swiss-Prot/UniProt Public databases

(Table 1) While a p-value of < 0.05 is generally

consid-ered significant for a specific function, each of the proteins

included in this study was highly significant for multiple

essential functions associated with angiogenesis (p = 10-4

to 10-12) (Table 1)

Approximately 88% (27 of 31) of the HIV-modulated

pro-teins could be located to the plasma membrane or

extra-cellular matrix of the infected cells (Figure 1) Functional

categorization of the identified proteins indicated that

each protein belonged to specific families of signal

trans-duction molecules including receptor or non-receptor

tyrosine kinases (ERBB2, ZAP70, FAK2), serine-threonine

kinases (KMLS, MAPK3 and PKC), lipid kinase (P3C2B/

PI3K), G-protein coupled receptors (BAI1, BAI3 and

CLR1), adhesion molecules/cytoskeletal proteins

(LAMA5, LAMB2, ITB5, FAT2, FINC), kinase adapters or

binding proteins (GRB2, CRKL and NELL1), protease/

peptidase (ATS9 and C3/CO3), regulatory enzyme

(NS2A), integral membrane proteins (TNR9 and GLG1),

calcium-binding protein (ANX-A6) and coagulation

fac-tor (VWF) (Figures 2 &3) Although numerous

transcrip-tion factors were induced de novo or upregulated

post-HIV-Infection of T-cells, in the present analysis, we have

considered the endothelial cell-specific zinc finger

tran-scription factor (ZNF71) induced by TNF alpha and

(TP53B), as important regulatory proteins that may be

necessary for the expression of cell-cycle genes/proteins

during the complex biological processes of angiogenesis

in vivo.

The VEGFR2 receptor and its growth factor ligand VEGFC

were downregulated in HIV-infected cells, although

detected only once in one of numerous acutely

HIV-infected cultures tested The PKC-regulatory protein 143G

was expressed at a lower level in HIV infected cells

com-pared to the uninfected controls The quantities of LAMA5and CLR1 were not much different between the infectedand uninfected cells (Figure 3) In addition a phosphatase(PPAC) was completely suppressed after HIV-infection(i.e detected only in the uninfected counterpart cells)(Figure 4) The downregulation of PPAC is considered to

be significant because its absence is essential for ing phosphorylation of various tyrosine kinases and acti-vation of endothelial cell growth in vivo [36]

maintain-The biological significance of all 31 proteins identified inthis study was computed in relation to protein-interactionnetworks involved in angiogenesis (p = 8 × 10-12) This, webelieve, is the first step toward developing a better insightinto the molecular mechanisms by which pathogenicviruses such as HIV may initiate and/or promote angio-genesis in the infected host

Stepwise Analyses of Essential Biological Processes in Angiogenesis

Angiogenesis is a multifactorial biological process ing numerous steps including endothelial cell activation,degradation of basement membrane, cell proliferation,invasion, morphogenesis, sprouting, migration and stabi-lization of microvessel formation Each step involves aseries of extremely complex but well-orchestrated protein-protein interactions along various signaling pathways Tounderstand the biological significance of each protein, wehave divided all proteins into 10 well-recognized biologi-cal events during neovascularization or angiogenesis(Table 1), and discussed putative functions of each pro-tein in that category Since most proteins are multifunc-tional, some overlap in the protein functions wasinevitable

involv-Step 1- Activation of T-Cells: Transcriptional and Translational Reprogramming

As soon as the HIV envelope glycoproteins (gp120/gp41)bind to the T-cell receptor and co-receptors (CD4, CXCR4and others), the cell surface proteins are clustered Thisgenerates a cascade of signals from the plasma membrane

to the cytoplasm and nucleus As the new proteins areexpressed, the HIV-infected cells are activated and aredriven toward apoptotic pathways [37,38] However,most activated cells also produce numerous cytokines,enzymes and other signal transduction molecules thatinvoke innate cellular immunity (to combat virus-infec-tion) and may be critical for the survival of the infectedcells These proteins maintain cellular integrity during var-ious phases of HIV replication and cell growth Many pro-

teins that are upregulated, downregulated or induced de

novo post-HIV infection may also be necessary to

compen-sate for the loss or disruption of essential physiological

functions performed by the T-lymphocytes prior to HIV

infection

Among a diverse family of multifunctional signaling

pro-teins induced de novo in HIV-infected cells, the protein

tyrosine kinases, the serine/threonine kinases and many

regulatory enzymes appear to play major roles in T-cell

activation and global reprogramming of the

transcrip-tional and translatranscrip-tional activities that lead to novel

inter-action pathways (Table 1)

Zeta Chain Tyrosine-Protein Kinase (ZAP-70)

The zeta chain protein tyrosine kinase (ZAP70-PTK) wasexpressed exclusively in HIV-infected cells (Table 1; Figure2) This kinase is associated with the zeta chain of the T-cell receptor (TCR) expressed on the plasma membrane.The tyrosine kinase activity of this receptor phosphor-

Table 1: HIV-Modulated Proteins Associated With Essential Steps During Angiogenesis

1 Activation of T-Cells: Transcriptional and Translational Reprogramming

2 Regulation of Cell Cycle: Lipid Kinase, Endothelial zinc finger and p53-binding protein

3 Augmentation of Cell Growth: Overexpression of Receptor Protein Tyrosine Kinases

4 Survival of Newly Formed Cells: Serine-Threonine Protein Kinase C (PKC) and Adapter Proteins

5 Mitogenic Signaling Cascade; Mitogen-activated Protein Kinase

6 Balanced Cell Growth or Adhesion: Anti-angiogenic G-Protein Coupled Receptors

7 Adhesion, Differentiation & Cell Migration: Focal Adhesion Kinase, Adhesion Receptor & Enzymes

8 Morphogenesis and Cell Migration: Laminins and other Cell Adhesion Molecules

9 Cell Permeability & Sprouting: Myosin Light Chain Kinase, Aggrecans & Peptidase

10 Preservation of Differentiated Cellular Phenotype: Coagulation-related Factor

HIV-modulated proteins significantly associated with essential biological steps in neovascularization and angiogenesis Four proteins were

upregulated, two were downregulated and all the rest (n = 25), were expressed de novo post-HIV-infection (i.e not expressed in uninfected

counterpart cells; Figures 1–4).

Since most of the proteins expressed in HIV-infected cells are multifunctional, the categorization of these proteins is only to facilitate a better understanding of numerous complex biological processes involved in angiogenesis Thus, PKC is listed in categories #1 and #4 and C3/C03 is listed

in #1 and #9

ylates multiple tyrosine residues of many functionally

important proteins (Figure 5) [39,40]

An important function of ZAP70 protein kinase in HIV

infected T-lymphocytes appears to be the suppression of

CD4-mediated CD3 signaling which selectively impairs

T-cell functions, reduces immune responses, induces anergy

and stimulates apoptosis in T-cells of both HIV-infected

and uninfected individuals [39] (p = 5 × 10-8) However,

in promonocytic cells, the HIV-encoded Nef protein

acti-vates the Src/Syk protein tyrosine kinase (SKF) activity

and recruits ZAP-70 [41] These multi-kinase complexes

have been reported to induce a cascade of signals which

cause downregulation of major histocompatibility

com-plex-1(MHC-I) via a membrane associated lipid kinase,

phosphatidylinositol-4-phosphate3-kinase C2-beta

(PI3K) pathway (Figures 2, 5), [41,42] Although this

interaction also affects immune evasion of HIV-infected

CD4+ T-cells, our experimentally-infected cells expressed

PI3K, concomitantly with the activation of ZAP-70 and

other protein tyrosine kinases Co-expression of these

pro-teins is critical for efficient coupling and antigen

recogni-tion of several intracellular signal transducrecogni-tion molecules

and may also promote cell-to-cell contacts and increased

HIV-spread [40,43]

An interesting finding relevant to our study was that the

upregulation of ZAP-70 PTK correlates negatively with the

expression of VEGF in patients with highly malignant,

angiogenic chronic B lymphocytic leukemia (CLL) [44,45].

Although B-cell functions are not compromised by anincrease in ZAP70 kinase, its expression on the surface ofCLL cells has been linked to the increased angiogenesisand poor prognosis of this cancer [45,46] On the con-

trary, absence of ZAP-70 expression was a good

prognosti-cator for CLL (i.e with less or no angiogenesis) althoughVEGF was expressed [44] These data suggest that VEGF-independent pathways were involved in CLL malignancy.Our proteomics and bioinformatics analyses of HIV-infected cells are consistent with these findings sinceexpression of ZAP-70 PTK and other PTK-containing pro-teins was associated with concomitant downregulation ofboth the VEGF and its cognate receptor VEGFR (p = 2.6 ×

10-3)

Tumor Necrosis Factor Receptor (TNR9)

One of the most frequently expressed cytokines during

HIV-infection in vitro or in vivo is the tumor necrosis factor

(TNF) The receptor for TNF belongs to the superfamily #

9 (TNR9) (synonyms: 4-1BB ligand receptor or CD137

antigen) was expressed de novo in the experimentally

HIV-infected cells (Table 1, Figure 2) This receptor is

impor-Cellular Locations of Differentially regulated Proteins in HIV-

Infected T-Cells

Figure 1

Cellular Locations of Differentially regulated

Pro-teins in HIV- Infected T-Cells The pie-chart illustrates

cellular localization of 31 proteins that were upregulated,

downregulated or induced de novo post-HIV infection

Pro-tein abbreviations are according to the Swiss-Prot/Uni-Prot

knowledgebase Asterisks (*) represent proteins that have

been primarily localized in the plasma membrane or

extracel-lular matrix but have been occasionally reported to be

expressed in the cytoplasm or other locations The

cytoplas-mic proteins include KMLS and MAPK3 (MKO3) and nuclear

proteins are TP53B and ZNF71 Full protein names,

abbrevi-ations and accession #s for each of all proteins are provided

in Table 1

Proteins Detected Exclusively in HIV-Infected Cells

Figure 2 Proteins Detected Exclusively in HIV-Infected Cells

Graph showing proteins that were detected exclusively in HIV-infected cells (i.e these proteins were not detected in counterpart uninfected cells at any time during the study) Although integrin (ITB5) was expressed in HIV-infected cells only, the small quantities could not be charted on the scale used X-axis shows protein abbreviations according to Swiss-PROT/UniProt databases Y-axis illustrates average of nor-malized quantity of specific protein spot computed automati-cally by the use of PDQuest program from multiple gels Error bars represent one standard deviation of the range for each protein data Full protein names, abbreviations and accession #s of each protein are provided in Table 1

0 1000 2000 3000 4000 5000 6000 7000 8000 9000

tant for the survival and maintenance of functional

changes in the CD4 and CD8 cells as immune effectors (p

= 8 × 10-8), [47]

The TNR9 receptor belongs to the TNF-nerve growth

fac-tor (NGF) recepfac-tor family and is activated by TNF or

related factors that are produced by most virus-infected

cells [48] Expression of TNR9 receptor facilitates

cluster-ing of T-cell receptors at the cell surface of HIV-infected

cells This interaction is conducive to activation of protein

kinases, and nuclear factor kappa B-associated signal

transduction pathways involved in the regulation of cell

growth, differentiation and inflammatory processes that

precede angiogenesis (p = 7 × 10-4), (Figure 5), [47-50]

Expression of TNR9 is also linked to the activation of

HIV-1 replication from latently infected CD4+ T cells [50,5HIV-1]

Upregulation of this receptor in HIV-infected cells may

therefore be essential for the sustained T-cell stimulation

and production of novel proteins that are needed to

facil-itate virus replication and synthesize virus particles

with-out killing the cell Although the expression of TNF has

been reported in many viral and microbial infections, the

upregulation of this factor in cancer cells has been

associ-ated with the induction of angiogenic factors [52]

Complement Receptor 3 (CO3/C3)

The complement receptor 3 (CO3/C3) was detected only

in HIV-infected cells (Table 1; Figure 2) This protein is thefirst responder of the innate immunity and is critical forthe protection of virus-infected hosts/cells Since aminoacid sequences of human C3 are similar to those of HIV-gp120 and gp41 envelope proteins, C3 can bind effi-ciently to different sites on the surface of T-cells and acti-vate them [53,54] Expression of C3 in HIV-infected cellsincreases the spread of virus to other cell types such asdendritic cells present in the peripheral blood of HIV-infected individuals [55-57]

One of the many critical functions of the C3 (and C5peptidases) is to stimulate chemotaxis and eventuallycontribute to the development of choroidal neovasculari-zation [58,59] These proteins also enhance permeability

of vasculature and cell migration during embryogenesis (p

= 4 × 10-4) Bioinformatics analyses indicates that a dinated expression of ZAP70, TNFR9 and C3, as well asthe release of these proteins in the blood of HIV-infectedindividuals, may be significantly involved in the initialgrowth and expansion of endothelial cells in early phases

coor-of angiogenesis (p = 7 × 10-4)

Protein Kinase C Beta Type (PKC)

Protein kinase C beta type (PKC) is a multifunctionalkinase, expressed exclusively in the HIV infected cells(Table 1; Figure 2) This kinase is essential for a wide range

of cellular functions including survival of activated T-cells

Proteins slightly Upregulated or same values

Post-HIV-infec-tion

Figure 3

Proteins slightly Upregulated or same values

Post-HIV-infection Graphic representation of two proteins

(LAMA5 and CLR1/CELSR1) showing approximately the

same values as control post-HIV-infection VWF was slightly

upregulated following HIV infection but was not statistically

significant in quantity FINC could not be charted because of

low levels X-axis = protein abbreviations are from

Swiss-PROT/UniProt Y-axis = average of normalized quantities of

proteins detected in multiple gels Error bars represent one

standard deviation for the range of each protein data Full

protein names and accession #s of each protein are provided

Proteins Down-regulated post-HIV- infection

Figure 4 Proteins Down-regulated post-HIV- infection Two

proteins were downregulated (1433G and PPAC) infection of T-cells X-axis = protein abbreviations according

post-HIV-to SwissPROT) Y-axis = average of normalized quantities of the same protein detected in multiple gels Error bars repre-sent one standard deviation for the range of each protein data Full protein names and accession #s of each protein are provided in Table 1

0 1000 2000 3000 4000 5000 6000 7000

(i.e protection of HIV-infected cells from apoptosis), cell

growth, and angiogenesis Presence of PKC induces many

intracellular signaling molecules that are not only critical

for the completion of virus life cycle [60,61], but are also

associated with T-cell activation and hyporesponsiveness

One of the first sets of signals generated in response to

extracellular stimuli involves the membrane-associated

lipid kinase phosphatidylinositol-4-phosphate3-kinase

C2-beta (PI3K or P3C2B) This kinase was induced de novo

in HIV-infected T-cells (Table 1; Figure 2) and is ered essential for the activation of these cells The PI3Kpreferentially phosphorylates phosphoinositide sub-strates that are necessary for cell cycle-related activities,DNA repair and cell proliferation [63,64]

consid-The expression of PI3K is necessary for many cal functions but the production of this lipid kinase may

physiologi-be enhanced by a variety of newly induced cytokines andthe HIV-encoded Tat protein expressed in the HIV-infected cells [64,65] Co-expression of PI3K with otherkinases discovered in this study may also be necessary forcell survival (i.e to keep the apoptotic pathways sup-

T-Cell Activation Pathways Generated by HIV-Modulated Proteins

Figure 5

T-Cell Activation Pathways Generated by HIV-Modulated Proteins Graphic representation of major proteins and

kinases involved in T-cell activation; the pathways were constructed by the direct Interaction Function Bioinformatics grams of Stratagene Pathway Architect 2.0.1 All proteins were uploaded and function-specific pathways were generated auto-matically; blue outlines around red ovals (ZAP 70, CRKL, and TNR9), indicate the activated proteins Note numerous cell surface proteins including PI3K involved in T-cell activation pathways Lines between red ovals denote major interactions; green circles represent small molecule interactions Full names of all protein abbreviations and accession numbers are listed in Table 1

Pro-pressed) in the HIV-infected T-cells and maintenance of

the overall health and metabolism of activated cells

dur-ing virus replication

Our bioinformatics analyses indicate that a coordinated

expression of PI3K with protein tyrosine kinases,

serine-threonine kinases and other signaling proteins in our

experimentally HIV-infected cells is critical for the

trolled growth of newly made endothelial cells Thus,

con-comitant expression of cell cycle genes, PI3K, MAPK and

FAK2 together with interacting partners ERBB2, GRB2 and

integrin v-beta (ITB5) in the HIV-infected T-cells is central

to the endothelial cell proliferation which is directly

rele-vant to various biological processes involved in

angiogen-esis PI3K is also recruited by a phosphotyrosine signaling

complex containing the activated receptor such as ERBB2

and a tyrosine kinase associated adapter protein GRB2

[66] Another important function of PI3K is its regulatory

role in the formation of tubular structures (vessels) during

angiogenesis [67], through a well-coordinated expression

of ITB5 and cell adhesion molecules that are crucial for

endothelial cell motility and intracellular signaling

path-ways (p = 2 × 10-5)

Endothelial Cell-Specific Transcription Factor, Zinc Finger (ZF71)

Although numerous transcription factors were

upregu-lated exclusively in our experimentally HIV-infected cells,

the activation of endothelial cell-specific zinc finger ZF71

(synonym: EZFIT) in T-cells is noteworthy (Table 1; Figure

2) This transcription factor mediates a wide range of

cel-lular functions such as transcriptional controls that

regu-late endothelial cell proliferation [68] The ZF71/EZFIT

mRNA levels were significantly upregulated when human

umbilical vein cells were treated with TNF-alpha [68] Our

bioinformatics analysis suggests that the upregulation of

TNR9, the receptor for TNF-alpha, and related factors in

HIV-infected T-cells may have enhanced the expression of

ZF71 Since TNF-alpha induces angiogenic factors in

can-cer cells [52] and upregulates production of signal

trans-duction molecules including chemokines [69], it is

probable that ZF71 promotes angiogenesis via the

expres-sion of tyrosine kinases and other critical enzymes in

HIV-infected cells

Tumor Suppressor p53-Binding Protein 1 (TP53B)

The tumor suppressor p53-binding protein 1 (TP53B or

53BP1) [70], was upregulated exclusively in HIV-infected

T-cells (Table 1; Figure 2) This is a highly conserved

nuclear protein associated with kinetochores

(microtu-bule attachment points associated with centromere) and

in some cells it shuttles between nucleus and cytoplasm

[71] Activation of this protein controls both the S phase

and G2/M phase checkpoint controls (p = 2.6 × 10-3)

Since TP53B also stimulates many different pathways

immediately after the double stranded DNA is perturbed

or damaged [71], it is likely that the integration of HIVprovirus in the cellular DNA may have triggered theexpression of cell-cycle-related pathways through TP53B.Our bioinformatics and statistical analyses indicate thatactivation of TP53B concomitantly with numerous upreg-ulated transcription factors, growth factors and enzymes

in HIV-infected cells, may be significantly associated withcell survival and growth (p = 2 × 10-4) Further, co-expres-sion of TP53B with the tyrosine kinase ERBB2, adhesionmolecules, LAMB2 and LAMA5, is also significantlyinvolved with the formation of vessels during embryonicdevelopment (p = 1.4 × 10-3)

Step 3- Augmentation of Cell Growth: Overexpression of Protein Tyrosine Kinases

The ERBB2 Receptor Protein Tyrosine Kinase

One of the most critical proteins induced by HIV appears

to be the ERBB2 receptor protein tyrosine kinase PTK; also known as HER-2/Neu or ERB2) (Table 1; Figure2) The ERBB2 protein was originally isolated as a viraloncoprotein, which belongs to the epidermal growth fac-tor (EGF) receptor family [72] This protein was notdetected in any of the numerous aliquots of the unin-fected T-cells tested at different stages of cell growth, over

(ERBB2-a period of two ye(ERBB2-ars Like most HIV-modul(ERBB2-ated proteinsidentified in the present study, expression of ERBB2 recep-tor has not been reported previously in HIV-infected cells.Since ERBB2-PTK shuttles back and forth from the cell sur-face to the nucleus [73], the intracellular "PTK-pool" inHIV-infected cells is enhanced due to phosphorylationand activation of numerous additional kinases, regulatoryenzymes, growth factors and other signaling proteins(Table 1, Figure 6 &7) The ERBB2 released in the circula-tion could therefore bind to cytokine-activated endothe-

lial cells in vivo and induce cell proliferative signals,

perhaps even before HIV has had a chance to replicate inthese cells

Expression of enhanced ERBB2 PTK activity has been ciated with highly malignant (angiogenic) ovarian andbreast cancers in women [74,75] Activation of ERBB2-

asso-PTK- receptor in human umbilical vein endothelial cells in

vitro stimulates proangiogenic factors independent of

VEGF-signaling [76] Studies in mouse cells have shownthat upregulation of ERBB2 transcription induces ang-iogenic factors while suppressing antiangiogenic factors[77]

Among the numerous functions of the ERBB2 receptor, its

involvement in the development of fetal endothelium[78]

is most relevant to the present study since 90% of ourHIV-induced proteins have been shown to be expressedduring the growth, neovascularization/angiogenesis and

development of the embryo The ERBB2 receptor is

acti-vated by a wide range of pleiotropic growth factors and

induces numerous signal transduction molecules which

stimulate endothelial cell growth during the development

of embryonic organs and angiogenesis [76,77] A

coordi-nated expression of ERBB2, with GRB2, PI3K, ZAP70 and

FAK-tyrosine kinase and other signaling proteins in the

experimentally HIV-infected cells is therefore anticipated

to activate multiple PTK- regulatory pathways, inhibit

apoptosis, enhance cell survival and stimulate endothelial

cell growth in vivo (p = 3 × 10 – 2 × 10-7) These results

indicate that predominant expression of

ERBB2-PTK-activity triggered solely by HIV-replication, without any

other intervention (infection or treatment), represents a

new dimension of VEGF-independent pathways involved

in neovascularization and angiogenesis (p = 4 × 10-4) Our

data also suggest that biological processes of angiogenesis

and embryonic development may be driven by common

pathways

Growth Factor Receptor-Bound Protein 2 (GRB2)

An important cell membrane-associated proteinexpressed in HIV-infected cells is the growth factor recep-

tor-bound protein 2 (GRB2) which interacts with the

acti-vated ERBB2 receptor PTK This protein is essential for the

transduction of growth-promoting signals involved inmorphogenesis as well as angiogenesis (Figures 6, 7) (p =

5 × 10-8)

GRB2 is associated with the activation of fetal genesthrough mitogen-activated protein kinase (MAPK) path-ways and is central to the functionalities of PI3K and othergrowth-stimulating kinases [79] that are also upregulated

by HIV-infection (Figure 4) Interaction of ERBB2 with theGRB2 protein is mediated by PI3K [66], while GRB2-asso-ciated scaffolding binding protein (GAB1) enhances cap-illary formation by coupling PI3K to VEGFR2 [80] Thecoupling properties of PI3K and the binding of GRB2 tothe activated ERBB2 in the presence of ZAP70-PTK and

Protein Interaction Pathways Involved in Augmentation of Cell Growth

Figure 6

Protein Interaction Pathways Involved in Augmentation of Cell Growth Cell growth-specific pathways were

con-structed by the direct Interaction Function Bioinformatics Programs of Stratagene Pathway Architect All proteins were uploaded and function-specific pathways were generated automatically Protein-protein- interactions involved in augmentation

of cell growth and angiogenesis along VEGF-independent pathways Note the VEGF-VEGFR interactions away from the

ERBB2-GRB2-MAPK3 (MKO3) Most of the regulatory proteins and kinases discovered in these pathways are normally expressed ing embryonic development Full names of all protein abbreviations and accession numbers are listed in Table 1

dur-other kinases is highly significant as these interactions

may not only stimulate endothelial cell growth along the

angiogenic pathways but also influence cell migration and

morphogenesis (p = 8 × 10-12), (Figures 6, 7)

Suppression of VEGF and its Cognate Receptor Tyrosine Kinase

The VEGF ligand and its cognate receptor VEGFR were not

detected in the experimentally HIV-infected T-cells tested

over a period of two years Only a single acutely-infected

culture showed basal levels of VEGF-C and its receptor

VEGFR-2 once and was not reproducible in duplicate wells

by MS The absence in HIV-infected cells was completely

unexpected since the HIV-encoded Tat binds VEGFR via

an arginine-glycine-aspartic-acid (RGD) region of

homol-ogy and activates angiogenic pathways through the PTK

activity of VEGFR [25,81] However, the RGD domains are

present in numerous integral plasma membrane proteinsidentified in this study including integrin and other celladhesion proteins [82] In addition, the binding of Tat toVEGFR is not as strong as the natural ligand (VEGF) and

the angioproliferative processes are triggered only when

Tat binds VEGFR in the presence of specific factors ing IL-1 beta, TNF-alpha, IFN-gamma or other angiogeniccytokines [8,81,83-85]

includ-As discussed above, our data has been corroborated byunrelated studies in which the expression of ZAP-70-PTK

suppresses VEGF expression [44] This fundamental

knowl-edge has provided new insights into the tyrosine signaling pathways likely to be generated by numerousPTKs, serine threonine kinases and other signaling pro-teins identified in the present study These mechanisms

kinase-Protein Tyrosine Kinase and other Major Kinases involved in Angiogenic Pathways

Figure 7

Protein Tyrosine Kinase and other Major Kinases involved in Angiogenic Pathways Pathways were constructed by

the direct Interaction Function Bioinformatics Programs of Stratagene Pathway Architect ALL proteins mapped in this figure

have been either upregulated or expressed de novo post-HIV-infection Proteins were uploaded and function-specific pathways

were generated automatically for protein tyrosine kinases expressed in HIV-infected cells Note that the newly discovered iogenic pathways involve distinct protein tyrosine kinases and signaling proteins as described in the text These pathways are

ang-independent of VEGFR2-VEGFC interactions as they do not interact with any of the proteins expressed in HIV-infected cells

Full names of all protein abbreviations and accession numbers are listed in Table 1